摘要
2-维凝胶电泳(Two-dimensionalgelelectrophoresis,2DE)因其高通量、高分辨率等特点,被广泛用于蛋白质组的分离.然而,在蛋白质从第一向-固相(ImmobilizedpHgradients,IPG)胶条转移到第二向-十二烷基磺酸钠聚丙烯酰胺凝胶(Sodiumdodecylsulfate,SDS;Polyacrylamidegelelectrophoresis,PAGE)时,常会引起蛋白质的损失.尤其是当将IPG胶条放入浓缩胶上时,在胶面不平、技术不熟练等情况下,常会在IPG胶条下引入气泡,导致蛋白质更多的损失.现将IPG胶条转移过程进行改进:先在第二向的十二烷基磺酸钠聚丙烯酰胺凝胶上加上薄薄的一层(约1～2mm厚)琼脂糖溶液,然后将IPG胶条转移上去,最后再封住胶条的上面.这样的转移不会引起气泡,可以大大提高实验的成功率及重复性,有利于蛋白质的转移(尤其是相对分子质量大于40kDa的蛋白质),提高质谱鉴定的准确性.此法操作简便,可以减少因操作不熟练而带来的麻烦.
Two dimensional gel electrophoresis (2DE) system is widely used for the separation of proteins because of its high throughput,high resolution and so on.However,there is usually enormous loss during the proteins transferred from the immobile IPG gel strip to the second dimensional gel procession.Sometimes when unskillful operator,or uneven gel surface results in some air bubbles under the IPG gel strip,there is more loss. The transfer method was improved in which immobile IPG gel was pressed to the stacking gel after adding a lamella hot agarose (about 1～2 mm) above the stacking gel,instead of pressed to the stacking gel straight.The improved transfer method will not induce air bubbles,so it can improve the chance of success and reproducibility,convenient for the transfer of proteins (especially of the higher molecular mass than 40 kDa proteins),and improve the accuracy of the identification by MS.Furthermore,the improved method is simple, it can decrease the trouble of unskillful operator.
出处
《生命科学研究》
CAS
CSCD
2004年第3期225-230,共6页
Life Science Research
基金
国家973基金资助项目(CBS-102)
关键词
2-维凝胶电泳(2DE)
蛋白质转移
改进
乳腺癌变组织
two dimensional gel electrophoresis (2DE)
protein transfer
improvement
carcinogenic mammary gland tissue
